The invention relates to methods of producing and characterizing compound libraries containing large numbers of multiply-substituted fullerenes. More particularly, the invention relates to chemically synthesizing combinatorial libraries of multiply-substituted fullerenes and to methods for efficiently screening for and identifying fullerene derivatives having pharmaceutical, materials science, or other utility. The invention also relates to the libraries thus produced, multiply-substituted fullerenes in the libraries which possess pharmaceutical, materials science, or other utility, and pharmaceutical formulations thereof.
The traditional method of generating compounds with desirable biological activity involves identifying a lead compound with the desired biological activity; creating, one at a time, variants of that lead compound; and evaluating the biological activity of those variants. Usually, these new medicinal chemical lead structures originate from natural products isolated from microbiological fermentations, plant extracts, and animal sources; from pharmaceutical company compound databases containing a historic collection of compounds synthesized in the course of pharmaceutical research; and from the application of both mechanism-based and structure-based approaches to rational drug design.
Accordingly, the traditional method of finding active pharmacological compounds requires the synthesis of individual compounds and the evaluation of their biological activity. Many hundreds of compounds are typically synthesized and screened before a substance with significant activity is identified which can serve as the lead structure for the development of drug candidates. Once a lead compound is found, analogs are synthesized to optimize biological activity. In addition to being a costly method of determining lead compounds, the traditional method of drug discovery has the additional disadvantage that one can never synthesize all of the possible analogs of a given, promising lead compound.
Recent trends in the search for biologically active compounds have focused on the use of combinatorial chemistry for the preparation of potential sources of new leads for drug discovery. Combinatorial chemistry is a strategy which leads to large chemical libraries. It is often defined as the systematic and repetitive, covalent connection of a set of different xe2x80x9cbuilding blocksxe2x80x9d of varying structures to each other to yield a large array of diverse, potentially pharmaceutically useful, molecular entities. Powerful techniques for the creation and screening of combinatorial libraries have been developed and improved upon rapidly in the past few years. These developments have rapidly expanded beyond their initial peptide and antibody targets to now include a wider range of biologically interesting compounds, as well as non-biological small molecules.
The libraries generated may each contain vast numbers of different molecules. Screening and isolation procedures are available which offer the means to identify and isolate compounds from a library which fulfill specific biological requirements. These methods include inhibition of binding of tritiated radioligands or selected fluorescence-labeled selected ligands to cell surface receptors on intact cells in culture, to cell surface receptors on disaggregated cell membranes, to cell surface receptors on cells in which a cloned neurotransmitter has been transfected, to cell surface receptors on tissue slices mounted upon microscope slides, to cell surface receptors on tissue strips maintained in organ baths, to cell surface receptors on whole organs maintained perfused and oxygenated in vitro, and to whole organs in the animal in vivo. The method also includes inhibition of binding of ligands to purified or cloned, recombinant receptors immobilized upon a chemical sensor, to purified or cloned, recombinant receptors immobilized upon an optical sensor, to purified or cloned, recombinant receptors immobilized upon an electromechanical sensor, and so forth. All of these techniques are well known to those skilled in the art.
The combinatorial chemistry approach does not actually change the medicinal chemistry paradigm. It introduces the new step of creating libraries, and accelerates the otherwise time consuming process of finding these compounds. By greatly increasing the range of molecular diversity available to the medicinal chemist, combinatorial chemistry has the potential to greatly broaden the number of molecules being surveyed for biological activity and other desirable properties.
The essential starting point for the generation of a diverse library of molecules is an assortment of small, reactive molecules which may be considered chemical building blocks. Unlike the traditional method, where the goal is to prepare and isolate individual variants of a lead compound, the combinatorial method deliberately creates a diverse set of variants simultaneously. The variants are then screened for useful properties.
Theoretically, the number of possible different individual compounds, N, prepared by an ideal combinatorial synthesis is determined by the number of blocks available for each step (xe2x80x9cbxe2x80x9d) and the number of synthetic steps in the reaction scheme (xe2x80x9cxxe2x80x9d). If an equal number of building blocks are used in each reaction step, then N=bx.
For example, it is well known in the art that multiple peptides and oligonucleotides may be simultaneously synthesized. In a single synthesis of a peptide, amino acids are simultaneously coupled to a chemically functionalized solid support. Typically, an N-protected form of the carboxyl terminal amino acid, e.g. a t-butoxycarbonyl protected (Boc-) amino acid, is reacted with the chloromethyl residue of a chloromethylated styrene divinylbenzene copolymer resin to produce a protected amino acyl derivative of the resin, the amino acid being coupled to the resin as a benzyl ester. This derivative is deprotected and reacted with a protected form of the next required amino acid thus producing a protected dipeptide attached to the resin. The amino acid will generally be used in activated form, e.g. a carbodiimide or active ester. The addition step is repeated and the peptide chain grows one residue at a time by condensation of the required N-protected amino acids at the amino terminus until the required peptide has been assembled on the resin. The peptide-resin is then treated with anhydrous hydrofluoric acid to cleave the ester linking the assembled peptide to the resin and liberate the required peptide. The protecting groups on side chain functional groups of amino acids which were blocked during the synthetic procedure, using conventional methods, may also be removed. This entire procedure may be automated. Multiple peptides or oligonucleotides may be synthesized.
One such methodology for peptide synthesis is disclosed in Geysen, et al. International Publication Number WO 90/09395, hereby incorporated by reference. Geysen""s method involves functionalizing the termini of polymeric rods and sequentially immersing the termini in solutions of individual amino acids. Geysen""s approach has proven to be impractical for commercial production of peptides since only very minute quantities of polypeptides may be generated. In addition, this method is extremely labor intensive.
U.S. Pat. No. 5,143,854 to Pirrung et al., hereby incorporated by reference, discloses another method of peptide or oligonucleotide synthesis. This method involves sequentially using light for illuminating a plurality of polymer sequences on a substrate and delivering reaction fluids to said substrate. A photochemical reaction takes place at the point where the light illuminates the substrates. Reaction at all other places on the substrate is prevented by masking them from the light. A wide range of photochemical reactions can be employed in this method, including addition, protection, deprotection, and so forth, as are well known in the art. This method of synthesis has numerous drawbacks, however, including the fact that the products are non-cleavable and that the process produces large numbers, but only minute quantities, of products.
A further method and device for producing peptides or oligonucleotides is disclosed in European Patent No. 196174. The disclosed apparatus is a polypropylene mesh container, similar to a tea-bag, which encloses reactive particles. The containers, however, are not amenable to general organic synthesis techniques.
Further apparatus are disclosed in German Published Patent Application No. DE 4005518 and European Patent No. 0355582. This apparatus is not suitable for the synthesis of general organic compounds is directed to peptide or oligonucleotide synthesis.
The synthesis of general organic compounds poses many difficulties which are absent in the synthesis of peptides or oligonucleotides. For example, it is difficult to provide a device which will accommodate the wide range of synthetic manipulations required for organic synthesis. The synthesis of general organic compounds often requires such varied conditions as an inert atmosphere, heating, cooling, agitation, and an environment to facilitate reflux. Additionally, such synthesis requires chemical compatibility between the materials used in the apparatus for multiple synthesis and the reactants and solvents. Consequently, the apparatus must be constructed of materials which are resistant to organic synthesis conditions and techniques. organic synthesis also often requires agitation. Such agitation may be accomplished by magnetic stirring, sonicating or rotational shaking. None of the prior art devices are suitable for use under these special conditions required for general organic synthesis.
Techniques have been developed in which libraries of organic compounds are synthesized on a solid support and screened for promising lead compounds. For example, U.S. Pat. No. 5,288,514 to Ellman et al., hereby incorporated by reference, describes the combinatorial synthesis of benzodiazepine compounds on a solid support. Solid phase syntheses have been found to be suitable for automation, and these chemical and biological methods have recently been refined for the generation of large combinatorial libraries that are screened against a specific receptor or enzyme in order to determine the key molecular recognition elements of the compounds for that receptor or enzyme.
While combinatorial synthesis of linear peptides or oligonucleotides is easier than synthesis of non-peptide organic compounds, peptides in general are not promising therapeutic agents. Their limited utility as bioavailable therapeutic agents is due to problems related to drug delivery and metabolism that are well known to those skilled in the art. For example, peptide therapeutics generally can only be administered by injection or inhalation, rather than orally, which is preferred for medications which are to be administered regularly outside of a doctor""s office. They also tend to have rapid clearing times. Furthermore, there remain major difficulties in targeting the peptide to the anatomical location where its action is desired.
For these reasons, there has been interest in the chemical synthesis of modified peptides, containing N-methylated backbones, peptide aldehydes, and peptide bonds replaced with methylene linkages, for example, which result in increased permeability through cell membranes and decreased metabolic destruction or destruction by enzymes. However, the synthesis of such modified peptides is expensive and complex, and the design of appropriate analogs to natural peptides frequently is far from straightforward. Further, the building blocks utilized are, in general, limited, even allowing for the use of unnatural enantiomers or artificial amino acids and modified nucleotides. The peptides or oligonucleotides generated possess a repetitive linkage through an amide or phosphate moiety, which limits their structural diversity.
The difficulties with peptides have created a need for small molecular templates suitable for substitution utilizing combinatorial methods to produce compounds with chemical, pharmaceutical and related utilities. of particular value are templates capable of producing compounds useful as drugs for the targeting of enzymes, regulatory proteins and cellular receptors.
Agonists and antagonists of various receptors having central nervous system (CNS) activity are of great interest. For example, adenosine receptor agonists and antagonists have a wide range of potential therapeutic utilities. In the cardiovascular system, A-2 agonists can increase coronary blood flow and can serve as peripheral vasodilators. A-2 agonists have been shown to possess antipsychotic activity in the appropriate preclinical animal models and can also have desirable sedative properties. More speculatively, adenosine receptor agonists may also be effective as antihypertensive agents, in the treatment of opiate withdrawal, as modulators of immune competence and renin release, antiasthmatics, and in the treatment of respiratory disorders.
Calcium channels are physiologically very important because they have a central role in regulating intracellular Ca2+ levels, which are vitally important for cell viability and function. Ca2+ functions in many ways as a hormone and second messenger. Ca2+ concentrations are implicated in the normal function of a number of vital processes, such as neurotransmitter release, muscle contraction, pacemaker activity, and secretion of hormones and other substances. A number of compounds useful in treating various diseases such as hypertension in animals, including humans, exert their beneficial effects by modulating functions of voltage-dependent calcium channels. It is well known that accumulation of calcium in the brain cells (calcium overload) is seen after periods of uncontrolled hyperactivity in the brain, such as after convulsions, migraine, anoxia and ischemia. As the concentration of calcium in the cells is of vital importance for the regulation of cell function, an uncontrolled high concentration of calcium will lead to the symptoms and possibly also the degenerative changes combined with the above diseases. Therefore, Ca2+ blockers selective for brain cells will be useful in the treatment of anoxia, traumatic injury, ischemia, migraine and epilepsy.
L-glutamic acid, L-aspartic acid and several other closely related amino acids have in common the ability to activate neurons in the central nervous system. Acidic amino acids are well known to be neurotransmitters for the vast majority of excitatory neurons. However, the excessive or inappropriate stimulation of excitatory amino acid receptors can lead to neuronal cell damage via a mechanism known as excitotoxicity. This process has been suggested to mediate neuronal degeneration in a plethora of disease processes. Therefore, the amelioration of these degenerative neurological processes is an important therapeutic goal.
Excitatory amino acids exert their actions through specific receptors located postsynaptically or presynaptically. Such ion-channel-linked receptors are subdivided into three groups based on electrophysiological and neurochemical evidence: the NMDA (N-methyl-D-aspartate) receptors, the quisqualate receptors, and the kainate receptors. L-glutamic acid and L-aspartic acid probably activate all of the three types of excitatory amino acid receptors.
In view of the limitations and shortcomings of the prior art, it is apparent that there still remains a need to provide small molecular templates suitable for substitution utilizing combinatorial methods. It is therefore an object of this invention to provide novel organic compounds with pharmaceutical, materials science or other utility.
It is another object of this invention to provide a method for preparing libraries of said compounds or their pharmaceutically acceptable salts.
A further object of this invention is to provide methods for identifying and isolating the members of said libraries.
Yet another object of this invention is to provide 20 a method for the screening of said libraries to determine the activity of the compounds therein and for the separation of the biologically active compounds in said libraries from the inactive compounds.
Still another object of this invention is to provide novel pharmaceutical and controlled-release compositions and methods for treatment utilizing said biologically active compounds.
Still another object of this invention is to provide methods for using the compounds of said libraries for diagnostic purposes and in biosensors.
Additional objects and advantages of the invention will be set forth in the description that follows.
The invention is directed to a method for the preparation and screening, preferably in parallel and simultaneous fashion, of large numbers of multiply-substituted fullerene derivatives. A method is provided for the preparation of combinatorial libraries of multiply-substituted fullerene compounds, some of which compounds possess pharmaceutical, materials science, or other utilities.
The invention also relates to novel multiply-substituted fullerenes which possess useful biological activity and to pharmaceutical formulations thereof, as well as precursors for making them. These multiply-substituted fullerenes have the general structure of formula I: 
wherein:
Z1, Z2 and Z3 are absent or present, provided that at least one is present, and are independently selected from the group consisting of xe2x80x94CR1R2xe2x80x94, xe2x80x94CR1R2-CR3R4xe2x80x94, xe2x80x94NR1xe2x80x94, xe2x80x94Oxe2x80x94CR1R2xe2x80x94, xe2x80x94Sxe2x80x94CR1R2xe2x80x94, xe2x80x94NR1xe2x80x94CR2R3xe2x80x94, xe2x80x94R1R2Cxe2x80x94NR3xe2x80x94CR4R5xe2x80x94, xe2x80x94R1Cxe2x95x90Nxe2x80x94CR2R3xe2x80x94, xe2x80x94R1R2Cxe2x80x94NR3xe2x80x94NR4xe2x80x94, xe2x80x94R1R2Cxe2x80x94NR3xe2x80x94Oxe2x80x94, xe2x80x94Nxe2x95x90NR1xe2x80x94, xe2x80x94Nxe2x95x90Nxe2x80x94NR1xe2x80x94, xe2x80x94Nxe2x95x90Nxe2x80x94CR1R2xe2x80x94, xe2x80x94Oxe2x80x94NR1xe2x80x94Oxe2x80x94, xe2x80x94R1R2Cxe2x80x94Oxe2x80x94CR3R4xe2x80x94, xe2x80x94R1R2Cxe2x80x94Oxe2x80x94NR3xe2x80x94, xe2x80x94R1Cxe2x95x90Nxe2x80x94NR2xe2x80x94, xe2x80x94R1Cxe2x95x90Nxe2x80x94Oxe2x80x94, xe2x80x94R1Nxe2x80x94NR2xe2x80x94NR3xe2x80x94, xe2x80x94R1Nxe2x80x94NR2xe2x80x94Oxe2x80x94, xe2x80x94CR1R2xe2x80x94CR3R4xe2x80x94CR5R6xe2x80x94, xe2x80x94CR1R2xe2x80x94CR3xe2x95x90CR4xe2x80x94, xe2x80x94CR1R2xe2x80x94CR3R4xe2x80x94CR5CR6xe2x80x94CR7R6xe2x80x94, xe2x80x94CR1xe2x95x90R2xe2x80x94CR3R4xe2x80x94CR5R6xe2x80x94, xe2x80x94CR1R2xe2x80x94CR3xe2x95x90CR4xe2x80x94CR5R6xe2x80x94, xe2x80x94CR1xe2x95x90CR2xe2x80x94CR3xe2x95x90CR4xe2x80x94 and xe2x80x94CR1R2xe2x80x94CR3xe2x95x90C1⊚CR4xe2x80x94 such that Z1, Z2 and Z3 are each attached to the carbon skeleton of the fullerene structure by two single bonds selected from the group consisting of Cxe2x80x94C, Cxe2x80x94O, Cxe2x80x94S or Cxe2x80x94N, the unsatisfied valences of each Z moiety being the location of those bonds; R1, R2, R3, R4, R5, R6, R7 and R8 may be the same or different and are selected from the group consisting of hydrogen, oxygen, lower alkyl, higher alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, carboxylic acids, carboxylic esters, alkylthio, thioalkyl, aryl, aryloxy, aralkyl, primary amine, secondary amine, amino acid side chains, and heterocycles, such that C together with any two R groups bonded thereto may form an oxo or thioxo group, hydrocarbon ring or heterocycle;
Y1 and Y2 are absent or present, provided that at least one is present, and are selected from the group consisting of hydrogen, lower alkyl, higher alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, alkylthio, thioalkyl, aryl, aryloxy, aralkyl, primary amine, secondary amine, amino acid side chains, and heterocycles;
20 less than n less than 240;
(k1+k2) is 1 to n; and
(m1+m2+m3) is 1 to n/2, with the limitation that 2(m1+m2+m3)+(k1+k2)xe2x89xa6n;
or a salt or addition compound thereof;
or the general structure of formula II: 
wherein:
Z4, Z5 and Z6 are absent or present, provided that at least two are present, and are independently selected from the group consisting of xe2x80x94CR9R10xe2x80x94, xe2x80x94CR9R10xe2x80x94CR11R12xe2x80x94, xe2x80x94NR9xe2x80x94, xe2x80x94Oxe2x80x94CR9R10xe2x80x94, xe2x80x94Sxe2x80x94CR9R10xe2x80x94, xe2x80x94NR9xe2x80x94CR10R11xe2x80x94, xe2x80x94R9R10Cxe2x80x94NR11xe2x80x94CR12R13xe2x80x94, xe2x80x94R9Cxe2x95x90Nxe2x80x94CR10R11xe2x80x94, xe2x80x94R9R10Cxe2x80x94NR11xe2x80x94NR12xe2x80x94, xe2x80x94R9R10Cxe2x80x94NR11xe2x80x94Oxe2x80x94, xe2x80x94Nxe2x95x90NR9xe2x80x94, xe2x80x94Nxe2x95x90Nxe2x80x94NR9xe2x80x94, xe2x80x94Nxe2x95x90Nxe2x80x94CR9R10xe2x80x94, xe2x80x94Oxe2x80x94NR9xe2x80x94Oxe2x80x94, xe2x80x94R9R10Cxe2x80x94Oxe2x80x94CR11R12xe2x80x94, xe2x80x94R9R10Cxe2x80x94Oxe2x80x94NR11xe2x80x94, xe2x80x94R9Cxe2x95x90Nxe2x80x94NR10xe2x80x94, xe2x80x94R9Cxe2x95x90Nxe2x80x94Oxe2x80x94, xe2x80x94R9Nxe2x80x94NR10xe2x80x94NR11xe2x80x94, xe2x80x94R9Nxe2x80x94NR10xe2x80x94Oxe2x80x94, xe2x80x94CR9R10xe2x80x94CR11R12xe2x80x94CR13R14xe2x80x94, xe2x80x94CR9R10xe2x80x94CR11xe2x95x90CR12xe2x80x94, xe2x80x94CR9R10xe2x80x94CR11R12xe2x80x94CR13CR14xe2x80x94CR15R16xe2x80x94, xe2x80x94CR9xe2x95x90R10xe2x80x94CR11R12xe2x80x94CR13R14xe2x80x94, xe2x80x94CR9R10xe2x80x94CR11xe2x95x90CR12xe2x80x94CR13R14xe2x80x94, xe2x80x94CR9xe2x95x90CR10xe2x80x94CR11xe2x95x90CR12xe2x80x94 and xe2x80x94CR9R10xe2x80x94CR11xe2x95x90Cxe2x95x90CR12xe2x80x94, such that Z4, Z5 and Z6 are each attached to the carbon skeleton of the fullerene structure by two single bonds selected from the group consisting of Cxe2x80x94C, Cxe2x80x94O, Cxe2x80x94S or Cxe2x80x94N, the unsatisfied valences of each Z moiety being the location of those bonds;
R9, R10, R11, R12, R13, R14, R15 and R16 may be the same or different and are selected from the group consisting of hydrogen, oxygen, lower alkyl, higher alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, carboxylic acids, carboxylic esters, alkylthio, thioalkyl, aryl, aryloxy, aralkyl, primary amine, secondary amine, amino acid side chains, and heterocycles, such that C together with any two R groups bonded thereto may form an oxo or thioxo group, hydrocarbon ring or heterocycle;
20 less than n less than 240; and
(m4+m5+m6) is 2 to n/2;
or a salt or addition compound thereof;
or the general structure of formula III: 
wherein:
V, W and X are absent or present, provided that at least two are present, and are selected from the group consisting of hydrogen, lower alkyl, higher alkyl, cycloalkyl, alkenyl, alkynyl, carboxylic acids, carboxylic esters, alkoxy, alkylthio, thioalkyl, aryl, aryloxy, aralkyl, primary amine, secondary amine, amino acid side chains, and heterocycles;
20 less than n less than 240; and
(a+b+c) is 1 to n/2;
or a salt or addition compound thereof. Preferably n is 60, 70 or a mixture of 60 and 70.
Preferred specific compounds include those compounds wherein the substituents contain functional groups which are amino acid side chains, or analogs of amino acid side chains.
The preparation of libraries of multiply-substituted fullerene libraries may result in the preparation of compounds which possess pharmaceutical, materials science, or other utility. Certain of the fullerene derivatives may be useful as drugs for the targeting of enzymes, regulatory proteins and receptors of various kinds. In particular, certain multiply-substituted fullerenes of the claimed invention may be used in pharmaceutical compositions for the treatment of various central nervous system, cardiovascular and respiratory disorders. Multiply-substituted fullerenes may also be used to form compositions for the controlled release of fragrances, pigments, moisturizers and other small molecules.
Other fullerene derivatives may be useful for, e.g., (i) the construction of batteries and similar devices such as fuel cells with improved electrochemical properties yielding typically increased storage times and at elevated currents; (ii) the construction of semiconductor devices such as diodes, transistors, field-effect devices, Josephson devices, superconducting quantum interference devices, electro-optically emissive diodes, transistors, and current-injection devices, and the like; (iii) the construction of electro-luminescent display devices such as flat-screen displays; (iv) the construction of electrical, optical, mechanical, magnetic, curie-point, or similar memory-storage devices such as are used in digital computers for the storage of binary information, including holographic or other optical-transform memory storage techniques; (v) the formulation, compounding, production, machining, and packaging of materials with superconductive properties; (vi) the formulation, compounding, production, machining, and packaging of materials with useful mechanical properties, typified by shear strength, Young""s modulus, ductility or other Theological characteristics, bulk modulus, lowered coefficient of friction in contact with any other material, and so forth; (vii) the formulation, compounding, production, machining, and packaging of materials with useful thermal properties, such as high thermal conductivity; (viii) the formulation, compounding, production, and packaging of materials with useful magnetic properties such as paramagnetism, diamagnetism, high or low magnetic susceptibility, unusual Curie point or Neel temperature, and the like. Materials science properties of fullerenes and fullerene deviations are based on unusual redox properties and the ability of metal salts to display conductivity and superconductivity.
In another embodiment, a method is provided for screening the libraries for active compounds which comprises 5 the steps of (a) contacting multiply-substituted fullerene compounds from the library with a biological target of interest, and (b) separating compounds which interact with the biological target from the inactive compounds. In this embodiment, compound libraries are screened for biological 10 activity by means of receptor binding assays or in vitro physiometric assays. In the most preferred embodiment, solid phase receptor binding assays are performed using a cloned receptor.
In a further embodiment, a method is provided for the separation of the active compounds from the inactive compounds. According to this method, libraries of multiply-substituted fullerenes can be indexed spectroscopically so that a desired compound can be identified and isolated from the library.
The invention also relates to the use of labelled fullerenes with the ability to target tumor cells as diagnostic agents, as well as the use of multiply-substituted fullerenes having biological activity in biosensors to detect analytes of interest.
The invention further relates to the preparation of monolayers and bilayers comprising multiply-substituted fullerenes in combination with a lipid.